Leydig cells are interstitial cells of the testis and represent the major source of testosterone (T) production in men. Normal T production is critical not only for normal sperm production and male fertility, but also for normal sexual behavior and male-specific physiology in general. The molecular mechanism underlying the development and function of Leydig cells remains largely undefined. We recently demonstrated in mice that when we inhibited the microRNA (miRNA) production by inactivating either of the two key RNase III enzymes, Dicer or Drosha, which are known to be essential for miRNA biosynthesis, Leydig cells could not function normally. In the absence of miRNAs, Leydig cells in adult mice overproduced testosterone, and the hypothalamus-pituitary-testis negative feedback system malfunctioned, leading to persistently elevated levels of T, decreased levels of estradiol (E2), and slightly increased levels of both gonadotropins (FSH and LH). This abnormal hormonal profile is accompanied by sperm defects characterized by separation of sperm heads from tails, lack of motility and deformation of sperm heads, which led to complete infertility in male mutant mice. Our preliminary data suggest that normal miRNA production is essential for normal Leydig cell functions [i.e. normal T and E2 production and normal hypothalamus-pituitary- testis (HPT) axis feedback], normal sperm production and male fertility. We, therefore, propose this pilot study to answer two fundamental questions concerning Leydig cell miRNAs: 1) How many miRNAs are normally expressed in Leydig cells? To answer this question, we will define the miRNA transcriptome in control and miRNA-deficient Leydig cells using miRNA deep sequencing (miRNA-Seq). 2) How does depletion of miRNAs affect mRNA transcriptome and proteome in Leydig cells? To answer these questions, we will define the mRNA transcriptome and proteome in adult control and miRNA-deficient Leydig cells using mRNA-Seq and iTRAQ-based mass spectrometry (MS). Data from this study will allow us to establish correlations between dysregulated genes leading to abnormal steroidogenesis and the absence of critical Leydig cell miRNAs (end-point of this study), which will serve as the basis for a further study on the specific mechanisms underlying the control of Leydig cell functions by miRNAs (starting point of the future R01 proposal). Future investigation based upon this pilot study may lead to novel diagnostics and treatments for male infertility due to endocrine disorders, and miRNAs may represent a novel class of male contraceptive agents.